本文選題：炭疽芽胞桿菌 + BslA蛋白�。� 參考：《安徽大學(xué)》2012年碩士論文

【摘要】：炭疽病是由炭疽芽胞桿菌(Bacillus anthracis)引起的一種人畜共患烈性傳染病,主要通過(guò)皮膚、呼吸道和消化道感染動(dòng)物和人,嚴(yán)重危害人類和家畜的生命健康。大量研究表明,單一的PA組分疫苗并不能夠提供完全的免疫保護(hù),以PA為主,輔以其它來(lái)自菌體或芽胞的保護(hù)性組份是一個(gè)比較理想的炭疽疫苗研究策略,因此研究者一直致力于尋找和鑒定新的炭疽芽胞桿菌保護(hù)性抗原。 炭疽芽孢桿菌的細(xì)胞外膜由一層二維晶狀結(jié)構(gòu)S層蛋白包裹,其生物學(xué)功能主要是作為保護(hù)性的外膜、分子泵和細(xì)菌粘附等。BslA蛋白(B. anthracis S-layer protein A)由炭疽芽胞桿菌pXO1致病島區(qū)域上的bslA(pXO1-90)基因編碼,包含652個(gè)氨基酸殘基。BslA蛋白N端1-259位氨基酸殘基中含有三個(gè)保守的SLH基序(S-layer-homologous motifs),是一種典型的S層蛋白。BslA蛋白炭疽芽胞桿菌感染宿主后有穩(wěn)定的表達(dá),天然表達(dá)的BslA與其它S層蛋白共同展示于菌體表面。研究表明BslA蛋白在炭疽芽胞桿菌致病過(guò)程中起著重要作用。本研究擬對(duì)BslA蛋白功能及其核心結(jié)構(gòu)域方面進(jìn)行研究,具體如下： 第一部分工作利用PCR的技術(shù)擴(kuò)增bslA基因片段,進(jìn)行原核表達(dá)載體構(gòu)建、重組蛋白分離純化、多克隆抗體制備,并且通過(guò)流式細(xì)胞、細(xì)菌粘附抑制實(shí)驗(yàn)、免疫熒光等實(shí)驗(yàn)方法鑒定BslA蛋白的功能。從實(shí)驗(yàn)結(jié)果來(lái)看,成功構(gòu)建重組菌株pET28a-BslA/Rosetta((DE3)并對(duì)BslA實(shí)現(xiàn)了可溶性表達(dá)。通過(guò)ProBondTM Purification system得到純度在87%以上的BslA蛋白,流式細(xì)胞與免疫熒光的結(jié)果表明BslA蛋白具有黏附功能。細(xì)菌粘附抑制實(shí)驗(yàn)計(jì)算出BslA重組蛋白及其抗血清能夠抑制約為98%的炭疽桿菌黏附,表明BslA蛋白可能是一種潛在的保護(hù)性抗原。 第二部分工作中,本實(shí)驗(yàn)通過(guò)蛋白結(jié)構(gòu)預(yù)測(cè)軟件,確定核心功能結(jié)構(gòu)域可能存在的位置,從而進(jìn)行分段研究。為了更好、更方便的檢測(cè),我們將蛋白片段與EGFP蛋白融合表達(dá),使EGFP成為標(biāo)簽。再通過(guò)免疫熒光等方法檢測(cè)(?)BslA蛋白片段功能。結(jié)果表明,成功構(gòu)建了pET28a-EGFP/Rosetta(DE3)和實(shí)現(xiàn)了可溶性表達(dá),并對(duì)其進(jìn)行了純化。表達(dá)分析和流式細(xì)胞檢測(cè)結(jié)果表明,構(gòu)建的EGFP蛋白有很好的熒光性。另外,根據(jù)蛋白結(jié)構(gòu)軟件的預(yù)測(cè)我們對(duì)BslA蛋白進(jìn)行了首次分段,成功構(gòu)建了pET28a-EGFP-BslA(260-500)/Rosetta(DE3)、pET28a-EGFP-BslA(411-560)/Rosetta(DE3)、pET28a-EGFP-BslA(511-652)/Rosetta(DE3),進(jìn)行了截短BslA的表達(dá)與純化。經(jīng)熒光電鏡檢測(cè)結(jié)果表明,BslA(260-500)、BslA(411-560)具有黏附功能,以此推斷核心功能結(jié)構(gòu)域位于BslA蛋白的411-500位氨基酸。根據(jù)此結(jié)果,進(jìn)行了第二次分段,成功構(gòu)建pET28a-EGFP-BslA(411-455)/Rosetta(DE3)、pET28a-EGFP-BslA(431-485)/Rosetta(DE3)、pET28a-EGFP-BslA(456-500/Rosetta(DE3)重組菌,對(duì)表達(dá)的蛋白進(jìn)行了純化。免疫熒光檢測(cè)的結(jié)果表明BslA(411-455)、BslA(431-485)片段具有粘附功能,進(jìn)一步確定了BslA蛋白的核心功能結(jié)構(gòu)域位于431455位點(diǎn)。 綜上,BslA是一種重要的保護(hù)性抗原,可能在新一代炭疽疫苗的研究和開發(fā)中扮演重要的角色。BslA蛋白是具有細(xì)胞粘附功能,其核心功能結(jié)構(gòu)域的確定為進(jìn)一步研究炭疽芽胞桿菌的致病機(jī)制與免疫預(yù)防打下了良好的基礎(chǔ)。
[Abstract]:Anthrax is a kind of zoonotic and zoonotic infectious disease caused by Bacillus anthracis (Bacillus anthracis), which mainly infect animals and people through skin, respiratory and digestive tract, and seriously endangers the life and health of human and domestic animals. A large number of studies have shown that a single PA vaccine can not provide complete immune protection, which is based on PA, supplemented by the vaccine. Other protective components from the bacteria or spore are an ideal research strategy for the anthrax vaccine, so researchers have been working to identify and identify new protective antigens of Bacillus anthracis.
The outer membrane of Bacillus anthracis is encapsulated by a two-dimensional crystalline structure of S layer protein. Its biological function is mainly as a protective outer membrane, and the.BslA protein (B. anthracis S-layer protein A) is encoded by the bslA (pXO1-90) gene on the region of Bacillus anthracis pXO1 pathogenicity island, including 652 amino acid residues. The 1-259 amino acid residues in the N terminal of the BslA protein contain three conservative SLH sequences (S-layer-homologous motifs). It is a typical S layer protein.BslA protein, Bacillus anthracis infected with the host, and has a stable expression. The natural BslA and other S layer proteins are displayed on the surface of the bacteria. The study shows that the BslA protein is in Bacillus anthracis. The purpose of this study is to study the function of BslA protein and its core domain, as follows:
In the first part, the bslA gene fragment was amplified by PCR technique, the prokaryotic expression vector was constructed, the recombinant protein was isolated and purified, and the polyclonal antibody was prepared. The function of BslA protein was identified by flow cytometry, bacterial adhesion inhibition experiment and immunofluorescence. From the experimental results, the recombinant strain pET28a-BslA/R was successfully constructed. Osetta (DE3) and the soluble expression of BslA were achieved. The purity of BslA protein above 87% was obtained by ProBondTM Purification system. The results of flow cytometry and immunofluorescence showed that BslA protein had adhesion function. The bacterial adhesion inhibition experiment calculated that the BslA recombinant protein and its antiserum could inhibit the adhesion of anthrax of about 98%. It is suggested that BslA protein may be a potential protective antigen.
In the second part, in this experiment, the possible location of the core functional domain was determined by the protein structure prediction software. In order to better, more convenient detection, we fused the protein fragment with EGFP protein to make EGFP a label. Then the function of BslA protein fragment was detected through immunofluorescence and other methods. The results showed that the soluble expression of pET28a-EGFP/Rosetta (DE3) was successfully constructed and purified. The results of expression analysis and flow cytometry showed that the constructed EGFP protein had good fluorescence. In addition, we successfully constructed the pET28a-EGFP based on the prediction of the protein structure software for the first time according to the protein structure software. The expression and purification of -BslA (260-500) (260-500) /Rosetta (DE3), pET28a-EGFP-BslA (411-560) /Rosetta (DE3), pET28a-EGFP-BslA (511-652) /Rosetta (DE3) were carried out. The results of fluorescence electron microscopy showed that BslA (260-500) and BslA (411-560) had adhesion function, so that the core functional domain was located in the 411-500 amino acids of the protein. According to the results, second subsections were carried out to successfully construct pET28a-EGFP-BslA (411-455) /Rosetta (DE3), pET28a-EGFP-BslA (431-485) /Rosetta (DE3) and pET28a-EGFP-BslA (456-500/Rosetta (DE3) recombinant bacteria to purify the expressed protein. The results of immunofluorescence detection showed that BslA (411-455), BslA (431-485) fragment had adhesion function. The core functional domain of BslA protein was located at the 431455 locus.
In conclusion, BslA is an important protective antigen. It may play an important role in the research and development of the new anthrax vaccine..BslA protein is a cell adhesion function. The determination of its core functional domain has laid a good foundation for further research on the pathogenesis and immune prevention of Bacillus anthracis.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378;S855.12

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